This invention relates to methods and apparatus in which target areas are illuminated with an array of spots or lines of light having very small dimensions.
Typical optical microscopy, far-field light microscopy, cannot resolve distances less than the Rayleigh limit. The Rayleigh criterion states that two images are regarded as just resolved when the principal maximum (of the Fraunhofer diffraction pattern) of one coincides with the first minimum of the other [see Born, M. and Wolf, E. Principles of Optics. Cambridge University Press 6th ed. p.415 (1980)]. For a circular aperture, this occurs at   w  =      0.61    ⁢          λ      NA      
For example, the wavelength (xcex) at the peak emission of a green fluorescent protein (EGFP) is 508 nm. Hence, for a very high numerical aperture (NA) of the objective, NA of 1.4, the minimum separation (w) that can be resolved in a GFP labeled sample is 221 nm. Currently, there are several possible methods for achieving resolution of spatial locations of proteins below the Rayleigh limit. They include: Confocal Microscopy, Fluorescence Resonance Energy Transfer (FRET), Atomic Force Microscopy (AFM), Near-Field Scanning Optical Microscopy (NSOM), Harmonic Excitation Light-Microscopy (HELM), Stimulated Emission Depletion Microscopy (STED-Microscopy) and Electron Microscope Immunocytochemistry.
Confocal Microscopy is a technique in which a very small aperture(s) is/are placed in the optical path to eliminate any unfocused light. This allows for a substantial increase in signal to noise ratio over conventional light microscopy. Also, it is possible to reduce the width of the central maximum of the Fraunhoffer pattern using a small slit or aperture. This, in turn allows a substantially enhanced resolution of 1.4 times better than the Rayleigh limit. Therefore, with this method, using the above protein as an example, a spatial resolution of 156 nm is achieved.
Typical confocal microscopy is not without disadvantages. By increasing the signal to noise ratio by decreasing the aperture size, the total signal level decreases concurrently. To bring the signal back to a useful level, the input power level must be increased. This, in turn, not only can cause photo-bleaching in the fluorophores at which one intends to look but also the surrounding area where the light is incident, just not collected. A method around this is to use two-photon excitation. Fluorescence from the two-photon effect depends on the square of the incident light intensity, which in turn, decreases approximately as the square of the distance from the focus. Because of this highly nonlinear (xcx9cfourth power) behavior, only those dye molecules very near the focus of the beam are excited, while the surrounding material is bombarded only by comparatively much fewer of the low energy photons, which are not of enough energy to cause photo bleaching. Multi-photon excitation requires highly skilled technicians and is somewhat expensive for clinical use. Because it acquires only a small area at once, the surface must be scanned in three dimensions for mapping.
Fluorescence Resonance Energy Transfer (FRET) can provide exquisite resolution of single chromophores. The resonance occurs when one fluorophore in an excited state transfers a portion of its energy to a neighboring chromophore. For this to take place, there must exist some overlap between the emission spectrum of the fluorophore to absorption spectrum of the chromophore (the frequency of the emission spectrum should be somewhat higher than the absorption spectrum of the chromophore). The process does not occur through photonic emission and absorption but through a dipole-dipole interaction. The strength of the interaction varies as rxe2x88x926. The Forster distance [see Forster, T Discuss. Faraday Soc. 27 7-29 (1959)] is the distance at which the efficiency of the transfer is such that there exists equal probability that the fluorophore loses energy to radiative decay or dipole-dipole interaction. The Forster distance, essentially, is the threshold at which FRET will no longer exist for a given pair. Typically the Forster distance is between 3 and 6 nm [see Pollok and Heim xe2x80x9cUsing GFP in FRET-based Applicationsxe2x80x9d Trends in Cell Biology 9 pp57-60 (1999)].
By placing either of the complementary pair near the other, resolutions of less than the Forster distance can be attained. The problem with this technique in determining relative locations is that one of the pair needs to be located within the resolution tolerances desired for spatial mapping. This can be achieved by placing one of the pair on a probe used in either atomic force microscopy (AFM) or near-field scanning optical microscopy (NSOM). Another problem is that dipole-dipole interactions are dependent on the relative orientation of the two. To maximize signal from the interaction would require a 3D scan around one of the pair.
Atomic Force Microscopy (AFM) can be envisioned as a very small (usually metal) stylus dragged across a surface giving feedback as to the height, Z, of the stylus relative to the surface. Resolution can be as fine as the scanning step size (typically 5 nm). By scanning across the surface, X and Y coordinates are obtained provided that the origin remains fixed (i.e., that there is no drift in the translation stage due to thermal or other effects). There are many methods for ensuring that the stylus does not actually contact the sample but maintains very accurate resolution of the Z coordinate. Because only surface morphology is measured, differentiating several molecules can be extremely difficult unless the dimensions and orientations of those molecules are well known. A solution to this might be to add tags of discrete lengths or shapes, which could be bound indirectly to the molecules of interest. This method, however, would require that the tissue sample to be planar before the tags were bound to the surface.
To increase the information of AFM, one could use Near-Field Scanning Optical Microscopy (NSOM or SNOM). NSOM uses a principle similar to AFM in which a stylus is scanned over a surface providing topographical information. However, the stylus is a conductor of photons. By emitting light from the tip of the stylus, optical measurements such as fluorescence can be obtained. Most often, these styli are fiber probes that have tapered tips and then are plated with a conductive material (aluminum is most often chosen as its skin depth for optical radiation is quite low, xcx9c13 nm at 500 nm) with a small aperture where the coating is broken. [See Betzig and Trautman xe2x80x9cNear-Field Optics: Microscopy, Spectroscopy, and Surface Modification beyond the Diffraction Limitxe2x80x9d Science 257 pp189-195 (1992)]. Another approach is to use what are called xe2x80x9capertureless probesxe2x80x9d [see Sanchez, Novotny and Xie xe2x80x9cNear-Field Fluorescence Microscopy Based on Two-Photon Excitation with Metal Tipsxe2x80x9d Physical Review Letters Vol 82 20 pp 4014-4017 (1999)] where an evanescent wave is excited by bombardment with photons at the tip of a sharpened metal probe. Because the tip can be made very sharp (radii of 5 nm are achievable), resolutions can be correspondingly smaller. An associated problem with the xe2x80x9capertureless probesxe2x80x9d is that the probe generates a white light continuum, which significantly decreases the signal to noise ratio.
By making the diameter (assuming a circular geometry) of the emission portion of the tip of the stylus very small (smaller than resolution desired) and keeping the tip to sample distance less than that distance, so that the diffraction is small, a nanometric light source is available. This light source can be used to excite fluorescence in the sample. Because the size of the source is very small and the scanning increments are also very small, highly resolved information on spatial locations of the fluorophores can be gleaned by inspection in the far field. Alternatively, the probe can be used for collection, measuring fluorescence or reflection or even transmission from illumination from the other side of the sample.
Because the aperture size in a conventional probe is so much smaller than the wavelength of the excitation light and only an evanescent mode is supported resulting in very little light is transmitted through the aperture. Diffraction effects limit the effective collimated length from the aperture to less than diameter of the aperture. This, then, requires that the aperture be held below a maximum height above the surface of the sample. Ideally, a fixed height above the surface (usually less than 10 nm) is used for relative contrast measurements. The height of the aperture relative to the surface is kept constant by measuring the shear force on the tip of the probe or by optical methods and is modulated to maintain that height. For this reason, NSOM is particularly susceptible to vibrations and experimental work requires isolation platforms.
Scanning the surface takes a fair amount of time. Thermal drift in commercially available open and closed loop nanometric scanning stages is about 20-30 nm/min. [see Frohn, Knapp and Stemmer xe2x80x9cTrue optical resolution beyond the Rayleigh limit achieved by standing wave illuminationxe2x80x9d Proceedings of the National Academies of Science Vol. 97, 13 pp 7232-7236 (2000)]. This can be severely limiting if scanning time is more than a few tens of seconds and resolution less than 50 nm is desired. If the surface is scanned for several different types of molecules, the required time to investigate a single cell becomes far too large for use in a clinical setting and would require multiple homings of the scanning stage. An approach to diminishing the scanning time may be to scan with multiple probes concurrently. This approach would be limited to just a few probes as on a small (202 xcexcm2) surface, the relatively large size of the probes"" bodies would interfere mechanically with each other.
U.S. Pat. Nos. 5,973,316 and 6,052,238 issued to Ebbesen et al. of the NEC Research Institute, Inc. describe a NSOM device which employs an array of subwavelength apertures in a metallic film or thin metallic plate. Enhanced transmission through the apertures of the array is greater than the unit transmission of a single aperture and is believed to be due to the active participation of the metal film in which the aperture array is formed. In addition to enhancing transmission, the array of apertures reduces scanning time by increasing the number of nanometric light sources.
A second method for increasing the number of light sources illuminates the sample with a mesh-like interference pattern and by post processing of the images. In Harmonic Excitation Light Microscopy (HELM), a laser is split into four beams and two of those beams modulated to produce an extended two-dimensional interference field with closely spaced antinodes. By introducing the beams at an angle to the surface to be imaged, an effective offset in reciprocal space is produced around an origin. If four images are taken around this origin and one at the origin, it is possible to construct, with post processing, a smaller single antinode which acts as a nanometric light source. This process can result in a lateral resolving power of close to 100 nm or half of the Rayleigh distance for green light. Because only a few images are required to map an entire surface, the acquisition time is extremely short (around 1.6 s for a 25 xcexcmxc3x9725 xcexcm area with 100 nm resolution.) Due to the required precision in the location of the four images around the origin and the drift associated with the scanning stage, it is unlikely that the resolution will be dramatically increased.
Another new form of microscopy is that introduced by Klar et al. [see Klar, Jakobs, Dyba, Egner and Hell xe2x80x9cFluorescence microscopy with diffraction resolution barrierxe2x80x9d Proceedings of the National Academies of Science Vol 97 15 pp 8206-8210 (2000)] called Stimulated Emission Depletion (STED) Microscopy. STED microscopy is based on a method of quenching fluorescence by stimulated emission depletion reducing the fluorescing spot size. [See Hell and Wichmann xe2x80x9cBreaking the Diffraction Resolution Limit by Stimulated-Emission-Depletion Fluorescence Microscopyxe2x80x9d Opt. Lett 19 11 780-782 (1994); Lakowicz, Gryczynski, Bogdanov and Kusba. xe2x80x9cLight Quenching and Fluorescence Depolarization of Rhodamine-B and Applications of this Phenomenon to Biophysicsxe2x80x9d J. Phys. Chem. 98 1 334-342 (1994); Hell, S. W. Topics in Fluorescence Spectroscopy, ed. Lakowicz (Plenum Press, NY), Vol. 5, pp. 361-422; and Klar and Hell xe2x80x9cSubdiffraction resolution in far-field fluorescence microscopyxe2x80x9d Opt. Lett 24 14, 954-956 (1999)]. Fluorescence can be quenched by subjecting a fluorophore to light at the lower energy edge (red side) of its emission spectrum. This forces the fluorophore to a higher vibrational level of the ground state, which, by decay of that state prevents re-excitation. Fluorescence can be turned on, with an ordinary excitation source, and turned off, with the STED beam, at will. By introducing an interference pattern in the STED beam, a local set of maxima and minima can be created. If the maxima of the STED beam are overlaid onto the fluorescence induced by the excitation beam, the fluorescence is quenched. However, where the minima occur, fluorescence continues. The fluorescing spot size is controlled by the union of the minimum or minima of the STED beam and the maximum of the excitation beam. Because STED is nonlinear with intensity, the sharpness of the minimum, maximum transition can be effectively increased allowing a narrow, almost delta behavior to be displayed. This, however, can result in severe photo stress to the sample and, possibly, dual photon effects, causing competing modes in the area where quenching is desired. So far, resolution in the radial (X, Y) direction is around 100 nm, but there is no reason to expect that the resolution can""t be substantially improved. Once again, though, STED microscopy is a scanning type and will suffer from the same drawbacks all scanning instruments do, (e.g., thermal drift, vibration problems, registration of near field excitement with far field collection and scan time.)
The present invention contemplates a different technique to achieve sub-Rayleigh criterion resolution, which is here called xe2x80x9cSurface Plasmon Enhanced Illuminationxe2x80x9d (SPEI). SPEI is related to NSOM in that multiple nanometric light sources are created by subwavelength apertures. By applying the principles of the present invention, a significant reduction in the size of the area illuminated by each aperture is achieved, resulting in significantly improved resolution.
The present invention takes the form of methods and apparatus that employ novel physical structures to provide nanometric spot or line illumination. In accordance with the invention, one or more apertures are formed through a first planar conductive material. Each aperture (which may be either a hole or a slit) has at least one cross-sectional dimension which is less than the wavelength of light which is incident to the planar material. In accordance with a feature of the invention, the structure includes means for confining the electronic excitation induced in that portion of the planar surface near the end of the aperture from which the light exits.
The conductive plane that receives the incident light may be placed on one outer surface of a dielectric material through which the aperture passes. The dielectric material prevents excitation of large densities of surface plasmons from being induced in an exposed conductive surface adjacent to the aperture opening where the light exits the structure.
Alternatively, the sidewalls of the aperture may be conductive to conduct excitation currents and act as a pseudo-waveguide for the light traveling through the aperture. At the exit end of the aperture, the amount of exposed conductive material is limited to an area immediately surrounding the hole exit by a dielectric material, or by a groove cut into the surface of the conductive material at the exit plane to a depth at least equal to the skin depth of the induced excitation and of such width and spacing to prevent an unwanted resonance of surface plasmons in that surface.
Alternatively, the conductive plane that receives the incident light may take the form of a xe2x80x9cgood metalxe2x80x9d layer with a xe2x80x9cbad metalxe2x80x9d layer having significantly different dielectric properties being sandwiched between the good metal layer and a dielectric substrate. The bad metal layer is preferably opaque to the light to be emitted from the surface of the good metal and its resonance (as determined by its dielectric function, the surface roughness and the dielectric functions of the materials on either side of the bad metal layer) should be very different from the resonance of the xe2x80x9cgoodxe2x80x9d metal, such that at desired frequency, light transmitted is emitted only from the holes and not from the exit surface of the array. The insulating dielectric substrate ensures that there can be no surface plasmon excitation from the good metal layer through the light barrier. When a bad metal layer is used that is both opaque to light and has sufficiently different dielectric properties relative to the good metal to eliminate resonant coupling, the dielectric insulator may be eliminated.
The present invention substantially reduces, compared to an array of subwavelength apertures in a monometallic film such as those described by Ebbesen et al., the size of the area of illumination produced by each aperture using the combination of a metallic layer on which surface plasmons are induced by incident light and surface composed of a material of substantially different dielectric function, such as an insulator or a different metal, so that the excitation of the surface plasmons in the light emitting surface in the exit surface layer will be different than those excited in the metallic layer that is excited by the incident light, and only the light from the decaying resonant surface plasmons of the exit layer will emit from that surface. The photons associated with the resonance of the incident or upper surface will be constrained to exit from the hole itself or from the walls of the hole.
In accordance with the invention, the light barrier comprises an illuminated surface consisting of a continuous conductive metallic layer in combination with an exit layer having substantially different dielectric properties. One or more apertures through the barrier (one or more holes or slits) then form xe2x80x9cphotonic funnelsxe2x80x9d through the barrier.
The invention may advantageously take the form of an array of apertures (holes or slits) formed in structure consisting of a dielectric substrate coated with a conductive metal film on one or both surfaces, or by a thick metallic film, and which further incorporates means for confining the electronic surface excitation to an area immediately adjacent to the apertures where light exits the structure. The means for confining the electronic surface excitation preferably takes the form of a layer of material having dielectric properties that differ substantially from those of the illuminated metal layer, and may consist of a dielectric insulator, a xe2x80x9cbad metalxe2x80x9d having different dielectric properties, grooves or surface irregularities at the exit surface, or a combination of these. The structure which confines the electronic surface excitation restricts the size of the spot or line of illumination from each aperture, and the use of an array of aperatures, or an array of surface irregularities on the metal film, increases the intensity of the illumination from each aperture
The present invention may also be applied to advantage in an optical data storage device. Several arrangements may be devised for combining the hole array with some medium for data storage. A light source, such as a laser, may be directed onto the front surface of the hole array which collects and funnels the array of light onto an optical storage medium. The bit value stored at each position in the storage medium controls the propagation of light through the storage medium to an adjacent pixel position in a charge coupled device (CCD) or other area detectors. A translation mechanism effects movement of the storage medium relative to the hole array in incremental steps, with each step distance being equal to the aperture size. In an alternative arrangement, data may be represented by illumination levels, such as gray scale values or color levels, and optical means may be used in place of or to supplement the mechanical translation mechanism.
The well defined and highly concentrated areas of illumination created by using such a structure as a light source provide significant advantages in microscopy and in optical data storage devices. The confined illumination patterns produced in accordance with the invention may be used to construct a xe2x80x9cSurface Plasmon Enhanced Microscopexe2x80x9d (SPEM) exhibiting markedly improved resolution, to construct an optical data storage device capable of storing larger amounts of data in optical storage media with much higher data access rates than is achievable with current optical data storage devices, and to provide a high throughput photolithography technique that can be applied to advantage in semiconductor fabrication and patterning for self-assembly and biological applications.
These and other objects, features and advantages of the present invention may be better understood by considering the following detailed description of specific embodiments of the invention. In the course of this description, reference will frequently be made to the attached drawings.